Targeted Protein Degradation by Electrophilic PROTACs that Stereoselectively and Site-Specifically Engage DCAF1

Abstract

Targeted protein degradation induced by heterobifunctional compounds and molecular glues presents an exciting avenue for chemical probe and drug discovery. To date, small-molecule ligands have been discovered for only a limited number of E3 ligases, which is an important limiting factor for realizing the full potential of targeted protein degradation. We report herein the discovery by chemical proteomics of azetidine acrylamides that stereoselectively and site-specifically react with a cysteine (C1113) in the E3 ligase substrate receptor DCAF1. We demonstrate that the azetidine acrylamide ligands for DCAF1 can be developed into electrophilic proteolysis-targeting chimeras (PROTACs) that mediated targeted protein degradation in human cells. We show that this process is stereoselective and does not occur in cells expressing a C1113A mutant of DCAF1. Mechanistic studies indicate that only low fractional engagement of DCAF1 is required to support protein degradation by electrophilic PROTACs. These findings, taken together, demonstrate how the chemical proteomic analysis of stereochemically defined electrophilic compound sets can uncover ligandable sites on E3 ligases that support targeted protein degradation.

Proteomic discovery of chemical probes that perturb protein complexes in human cells

“…We exposed 22Rv1 cells to two structurally distinct sets of four stereoisomeric electrophilic compounds (azetidine acrylamides ( Figure 1C ) (Tao et al, 2022) and tryptoline acrylamides ( Figure 1D ) (Vinogradova et al ., 2020) ; 20 µM, 3 h treatment), which were constructed based on principles of diversity-oriented synthesis (DOS) (Schreiber Stuart, 2000) – specifically the use of sp 3 -rich, entropically constrained, densely functionalized, and stereochemically defined scaffolds. We previously found that both sets of compounds stereoselectively engaged cysteine residues on diverse classes of proteins in human T-cells (Tao et al ., 2022; Vinogradova et al ., 2020). We accordingly sought herein to identify, from cells treated with stereoisomeric electrophilic compounds, proteins that showed stereoselective shifts in SEC migration (size shifts), which could then be correlated with stereoselective changes in cysteine reactivity.…”

Rational Chemical Design of Molecular Glue Degraders

“…Another major question is whether we can further expand the strategies that we have employed here to identify chemical handles against other E3 ligases beyond RNF126 that can be used to rationally develop molecular glue degraders. Given previous successes in using covalent ligands and chemoproteomic platforms to develop novel E3 ligase recruiters for PROTAC applications, including RNF114, RNF4, DCAF16, DCAF11, FEM1B, and DCAF1 and the high degree of E3 ligase ligandability using covalent strategies, it would be of future interest to further expand on the efforts started here with an expanded library of covalent moieties that could potentially access additional E3 ligases 23,28,34,[42][43][44][45][46] .…”

Advancing Targeted Protein Degradation via Multiomics Profiling and Artificial Intelligence

“…Initial work has predominantly focused on cysteine-targeting, covalent fragments. − These approaches have the advantage that they not only inform on the liganded protein but also precisely reveal the covalently bound cysteine residue. Of relevance for TPD, this has recently identified fragments that covalently engage E3 ligases. ,− Encouragingly, as mentioned above, these fragments could successfully be incorporated into functional PROTACs capable of inducing the degradation of BRD4 and other POIs. Future research will reveal if, in general, hits from covalent fragment screens can be advanced toward drug-like ligands for PROTAC or MGDs.…”